Methods of preventing and reducing the severity of stress-associated conditions

ABSTRACT

The present invention provides a method of preventing or reducing the severity of a stress-associated condition in a subject by systemically administering to the subject an effective amount of brimonidine or a pharmaceutically acceptable salt, ester, amide, sterioisomer or racemic mixture thereof. Stress-associated conditions that can be treated according to a method of the invention include, but are not limited to, dyspepsia, tachycardias other than tachycardia associated with myocardial ischemia, panic attack, non-inflammatory dermatogical conditions, disorders of muscle contraction, sensory hypersensitivity associated with migraine, and behavioral disorders.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates generally to the sympathetic nervous systemand various stress-associated conditions and, in particular, to the α-2adrenergic agonist, brimonidine.

[0003] 2. Background Information

[0004] Conditions that are associated with or exacerbated by stress canbe mediated, at least in part, by the sympathetic nervous system. Suchstress-associated conditions include, without limitation,gastrointestinal disease; irritable bowel syndrome; dyspepsia;tachycardia; panic attack; insulin-resistance; type II diabetes;dermatogical conditions; disorders of muscle contraction such as tensiontype headache; sensory hypersensitivity associated with migraine such asnausea, photophobia and phonophobia; and stress-associated behavioraldisorders such as overeating and drug dependence.

[0005] Unfortunately, treatments for such stress-associated conditionshave generally been ineffective or unsatisfactory, for example, due tounwanted side-effects such as sedation. Thus, there is a need for novelmethods of preventing or reducing the severity of stress-associatedconditions. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

[0006] The present invention provides a method of preventing or reducingthe severity of a stress-associated condition in a subject bysystemically administering to the subject an effective amount ofbrimonidine or a pharmaceutically acceptable salt, ester, amide,sterioisomer or racemic mixture thereof, where the stress-associatedcondition is one of the following: gastrointestinal disease; irritablebowel syndrome; dyspepsia; tachycardia; panic attack;insulin-resistance; type II diabetes; a non-inflammatory dermatogicalcondition; a disorder of muscle contraction; sensory hypersensitivityassociated with migraine; or a stress-associated behavioral disorder.

[0007] In one embodiment, a method of the invention prevents or reducesthe severity of gastrointestinal disease. In other embodiments, a methodof the invention prevents or reduces the severity of irritable bowelsyndrome or dyspepsia. In another embodiment, a method of the inventionprevents or reduces the severity of tachycardia other than tachycardiaassociated with myocardial ischemia, for example, tachycardia associatedwith a pulmonary disorder. In a further embodiment, a method of theinvention prevents or reduces the severity of panic attack. In stillfurther embodiments, a method of the invention prevents or reduces theseverity of insulin-resistance, or prevents or reduces the severity oftype II diabetes. In yet a further embodiment, a method of the inventionprevents or reduces the severity of a non-inflammatory dermatologicalcondition. In other embodiments, a method of the invention prevents orreduces the severity of a disorder of muscle contraction such as adisorder of skeletal muscle contraction or a disorder of smooth musclecontraction, for example, a disorder of smooth muscle contractionassociated with cystitis or associated with non-bacterial prostatitis ora disorder of muscle contraction associated with tension type headache.In another embodiment, a method of the invention prevents or reduces theseverity of sensory hypersensitivity associated with migraine. In afurther embodiment, a method of the invention prevents or reduces theseverity of sensory hypersensitivity associated with a stress-associatedbehavioral disorder. In a method of the invention, an effective amountof brimonidine can be administered by any of a variety of methodsincluding, but not limited to, orally, topically, intravenously or via apatch.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 shows the tactile hypersensitivity observed with severaldistinct chemical models. Each experimental group included 5-6 wildtypemice. Tactile hypersensitivity was assessed as described below;sensitization scores determined every 5 minutes during the 35 minutemeasurement period were summed and calculated as the mean +/−SEM. Eachgroup was compared to a vehicle control using an unpaired two-tailedt-test (* p<0.01, ** p<0.001). (a) Spinal injection of the α-1 agonist,phenylephrine, induces tactile hypersensitivity in a dose dependentfashion. Phenylephrine (filled circle) was injected intrathecally atvarious doses. The α-1 antagonist, 5-MU (30 ug/kg i.p.; filled square)was administered 15 minutes prior to intrathecal administration of 30 ngphenylephrine. (b) Systemic phenylephrine induces tactilehypersensitivity in a dose dependent fashion. Phenylephrine (filledcircle) was injected intraperitoneally at various doses. The α-1antagonist, 5-MU (30 ug/kg i.p.; filled square) was administered 15minutes prior to administration of 30 ng/kg phenylephrine. (c) Spinalsulprostone, a selective EP₁/EP₃ agonist, induces chemical tactilehypersensitivity in a dose responsive fashion. Increasing doses ofsulprostone (filled circle) were injected intrathecally. An EP₁antagonist (100 ng i.t.; filled square) was injected 15 minutes prior toadministration of 200 ng sulprostone. (d) Spinal administration of NMDAinduces tactile hypersensitivity in a dose responsive fashion. NMDA(filled circle) was injected intrathecally at various doses. The NMDAantagonist, memantine (1 ug i.t.; (filled square), was injected 15minutes prior to administration of 100 ng NMDA.

[0009]FIG. 2 shows that the increased sympathetic tone of α-2A and α-2Cknockout mice enhances induction of tactile hypersensitivity by α-1receptor activation. Wildtype (filled circle), α-2A knockout (filledsquare), and α-2C knockout (filled triangle) mice were injectedintraperitoneally with increasing doses of phenylephrine and assayed fortactile hypersensitivity. α-2A knockout mice were pretreated with 50mg/kg i.p. guanethidine to cause a temporary chemical sympathectomy24-30 hours prior to an i.p. injection with phenylephrine (open square).Each group of mice consisted of 5-6 animals. The mean sensitizationscore and SEM were calculated and compared to a vehicle control groupusing an unpaired two-tailed t-test (* p<0.01, ** p<0.001).

[0010]FIG. 3 shows that the sympathetic nervous system enhancessulprostone-induced tactile hypersensitivity. Wildtype (filled circle),α-2A (filled square), and α-2C (filled triangle) knockout mice wereinjected intrathecally with increasing doses of sulprostone and assayedfor tactile hypersensitivity. α-2A knockout mice were pretreated withguanethidine (50 mg/kg i.p.) to cause a temporary chemical sympathectomy24 hours prior to an intrathecal sulprostone injection (open square).Each group of mice consisted of 5-6 animals. The mean sensitizationscore and SEM were calculated and compared to a vehicle control groupusing an unpaired two-tailed t-test (* p<0.01, ** p<0.001).

[0011]FIG. 4 shows that α-2 knockout mice do not exhibit alteredNMDA-induced tactile hypersensitivity. Wildtype (filled circle), α-2A(filled square), and α-2C (filled triangle) knockout mice were injectedintrathecally with increasing doses of NMDA. Each group of 5-6 mice wasscored for tactile hypersensitivity. The mean response and SEM werecalculated and compared to a vehicle control group using an unpairedtwo-tailed t-test (* p<0.01, ** p<0.001).

[0012]FIG. 5 shows that α-adrenergic agonists differ in alleviation ofsympathetically-enhanced sensory hypersensitivity. The response of 5-6mice per group was scored; the mean response and SEM were calculated asdescribed above. Each drug-treated group was compared to a vehiclecontrol group using an unpaired two-tailed t-test (* p<0.01, **p<0.001). (a) Spinal brimonidine and clonidine alleviate NMDA-inducedtactile hypersensitivity in wildtype mice. Mice were injectedintrathecally with DMSO vehicle or co-injected intrathecally with 100 ngNMDA and saline, 0.4 μg brimonidine (UK14304) or 1 pg clonidine. (b)Spinal brimonidine and clonidine alleviate sulprostone-induced tactilehypersensitivity in wildtype mice. Mice were injected intrathecally withDMSO vehicle or co-injected intrathecally with 200 ng sulprostone andsaline, 0.4 μg brimonidine (UK14304) or 0.4 μg clonidine. (c) Spinalbrimonidine and clonidine alleviate NMDA-induced tactilehypersensitivity in the α-2C knockout mice, but not in the α-2A knockoutmice. Mice were injected intrathecally with DMSO vehicle or coinjectedintrathecally with 100 ng NMDA and saline, 0.4 μg brimonidine (UK14304)or 1 μg clonidine. (d) Spinal brimonidine and clonidine differ in theirability to alleviate sulprostone-induced tactile hypersensitivity in theα-2C knockout mice. Mice were injected with DMSO vehicle or co-injectedintrathecally with 200 ng (α-2C knockout) or 30 ng (α-2A knockout)sulprostone and saline, 0.4 μg brimonidine (UK14304) or 0.4 μgclonidine. α−2 agonist analgesia is absent in the α-2A knockout mice;clonidine analgesia is also lost in the α-2C knockout mice.

[0013]FIG. 6 shows that brimonidine, but not clonidine or tizanidine,alleviates sulprostone-induced tactile hypersensitivity in the absenceof sedation. The dose-responsive anti-hypersensitive and sedativeeffects of three α-2 agonists (tizanidine, triangle; clonidine, square;and brimonidine, circle) were compared in models of sulprostone-inducedtactile hypersensitivity and locomotor activity, respectively. The meantotal sensitivity score and standard error of the mean was calculatedand indicated as a solid line (left axis). Locomotor activity relativeto vehicle-treated animals was expressed as a percentage, and thepercent sedation calculated as 100% minus the percent locomotor activityand indicated as a hatched line (right axis).

[0014]FIG. 7 shows variable α-2 vs. α-1 agonist selectivity inα-adrenergic agonists clonidine and brimonidine. Increasingconcentrations of phenylephrine (filled square), clonidine (filleddiamond), tizanidine (filled circle), dexmeditomidine (filled triangle)and brimonidine (filled inverted triangle) were tested for α-1 and α-2agonist activity using in vitro cell-based functional assays. (a, b)α-1A and α-1B agonist activity of α-adrenergic agonists. The increase inintracellular calcium in HEK cells stably expressing the bovine α-1Areceptor (a) or the hamster α-1B receptor (b) following addition ofvarious concentrations of α-adrenergic agonists was determined bymeasuring the change in fluorescence of a calcium-sensitive dye.Agonists were tested 6-15 times in triplicate, and the mean fluorescenceand SEM calculated at each concentration. Results from a typicalexperiment are shown. (c, d) α-2A and α-2C agonist activity ofα-adrenergic agonists. Inhibition of forskolin-induced cAMP accumulationin PC12 cells stably expressing the human α-2A receptor (c) or the humanα-2C receptor (d) following addition of various concentrations ofa-adrenergic agonists. Agonists were tested 3-5 times in triplicate, andthe mean % inhibition and SEM calculated at each concentration. Resultsfrom a typical experiment are shown.

[0015] (e) Co-administration of prazosin with clonidine restoresclonidine-mediated analgesia in α-2C knockout mice. Wildtype (open bars)and α-2C knockout (hatched bars) mice were injected with vehicle,prazosin (100 ng/kg i.p.), sulprostone (200 ng i.t.), clonidine (400 ngi.t.) or various combinations as indicated. The tactile hypersensitivityof 5-6 mice per group was scored, and the mean response and SEM wascalculated. Each drug-treated group was compared to a vehicle controlgroup using an unpaired two-tailed t-test (* p<0.01, ** p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

[0016] Adrenergic receptors mediate physiological responses to thecatecholamines, norephinephrine and epinephrine, and are members of thesuperfamily of G protein-coupled receptors having seven transmembranedomains. These receptors, which are divided pharmacologically into α-1,α-2 and β-adrenergic receptor types, are involved in diversephysiological functions including functions of the cardiovascular andcentral nervous systems. The α-adrenergic receptors mediate mostexcitatory functions: α-1 adrenergic receptors generally mediateresponses in the effector organ, while α-2 adrenergic receptors arelocated postsynaptically as well as presynaptically, where they regulaterelease of neurotransmitters. Agonists of α-2 adrenergic receptorscurrently are used clinically in the treatment of hypertension,glaucoma, spasticity, and attention-deficit disorder, in the suppressionof opiate withdrawal, and as adjuncts to general anesthesia.

[0017] α-2 adrenergic receptors are presently classified into threesubtypes based on their pharmacological and molecular characterization:α-2A/D (α-2A in human and α-2D in rat); α-2B; and α-2C (Bylund et al.,Pharmacol. Rev. 46:121-136 (1994); and Hein and Kobilka, Neuropharmacol.34:357-366 (1995)). The α-2A and α-2B subtypes can regulate arterialcontraction in some vascular beds, and the α-2A and α-2C subtypesmediate feedback inhibition of norepinephrine release from sympatheticnerve endings. The α-2A subtype also mediates many of the centraleffects of α-2 adrenergic agonists (Calzada and Artiñano, Pharmacol.Res. 44: 195-208 (2001); Hein et al., Ann. NY Acad. Science 881:265-271(1999); and Ruffolo (Ed.), α-Adrenoreceptors: Molecular Biology,Biochemistry and Pharmacology S. Karger Publisher's Inc. Farmington,Conn. (1991)).

[0018] Previous studies have shown that norepinephrine has a higheraffinity for the α-2C receptor (Ki=650 nM) than the α-2A receptor(Ki=5800 nM; Link et al., Mol. Pharm. 42:16-27 (1992)). Thus, theautoinhibitory action on norepinephrine release is mediated through theα-2C receptor at low concentrations of norepinephrine, and through theα-2A receptor at high concentrations of norepinephrine (Altman et al.,Mol. Pharm. 56:154-161 (1999)). As a result, feedback inhibition ofbasal norepinephrine release is mediated by the α-2C receptor, while theα-2A receptor mediates feedback inhibition of release under conditionsof high frequency stimulation (Hein et al., Ann. N.Y. Acad. Sci.881:265-271 (1999)). As disclosed herein in Example II, the α-2Cknockout mice, which have a decreased presynaptic inhibition ofsympathetic outflow under basal (or low frequency stimulation)conditions, are more sensitive to augmentation of α-1 receptor activitythrough phenylephrine treatment (see FIG. 2). Furthermore, as shownherein in FIG. 3, α-2A knockout mice are more sensitive tosulprostone-induced tactile hypersensitivity, while in α-2C knockoutmice, the sulprostone sensitivity is the same as that of wildtype mice.These results demonstrate that sulprostone treatment results in highfrequency sympathetic nerve stimulation, as evidenced by the fact thatonly α-2A knockout mice, which lack presynaptic inhibition of highfrequency sympathetic outflow, exhibit a decreased threshold tosulprostone-induced tactile hypersensitivity.

[0019] As further disclosed herein in Example III, brimonidine wasanalgesic in both wild type and α-2C knockout mice withsulprostone-induced tactile hypersensitivity. In contrast, clonidine wasanalgesic in wild type mice but not in α-2C knockout mice (compare FIGS.5b and d). As expected, neither clonidine nor brimonidine were analgesicin α-2A knockout mice, which lack the spinal α-2A adrenergic receptorwhich mediates analgesic activity. Thus, in α-2C knockout mice treatedwith sulprostone, which serve as a model for sympathetically-enhancedconditions, the pan-agonists brimonidine and clonidine have strikinglydifferent activities. Additional results disclosed herein demonstratethat, in wild type mice, brimonidine, but not other pan-agonists such astizanidine or clonidine, had analgesic activity without concomitantsedation (see FIG. 6). Furthermore, brimonidine was highly selective(more than 1000-fold) for α-2 adrenergic receptors as compared to α-1receptors in functional assays as compared to other pan-agonists such asclonidine and tizanidine, which exhibited less than 10-fold selectivity(see FIG. 7 and Table 2). These results demonstrate the differentialfunctional activity of the pan-agonists brimonidine and clonidine andindicate that α-2 versus α-1 functional selectivity can be advantageousin treating sympathetically-enhanced conditions such asstress-associated conditions without concomitant sedation.

[0020] Dyspepsia has been described as a biopsychosocial disorder and isgenerally characterized, in part, by epigastric discomfort followingmeals. In addition to postprandial upper abdominal discomfort or pain,dyspepsia can be characterized by early satiety, nausea, vomiting,abdominal distension, bloating, or anorexia in the absence of organicdisease (Thumshirn, Gut 51 Suppl. 1: i63-66 (2002; Anderson, Dorland'sIllustrated Medical Dictionary 28^(th) Edition, W. B. Saunder's Company,Philadelphia (1994)).

[0021] The methods of the invention can be useful for preventing orreducing the severity of dyspepsia, which, as used herein, is a termwhich means impaired digestion. Any of a variety of types of dyspepsiacan be treated according to a method of the invention. The termdyspepsia includes, without limitation, acid dyspepsia, which isassociated with excessive acidity of the stomach; appendiculardyspepsia, also known as appendix dyspepsia, in which dyspeptic symptomsaccompany chronic appendicitis; catarrhal dyspepsia, which isaccompanied by gastric inflammation; chichiko dyspepsia, a condition offarinaceous malnutrition found in poorly nourished infants; cholelithicdyspepsia, which involves sudden dyspeptic attacks associated withgallbladder disturbance; colonic dyspepsia, which involves a functionaldisturbance of the large intestine; fermentive dyspepsia, which ischaracterized by fermentation of ingested food; flatulent dyspepsia,which is associated with the formation of gas in the stomach and ofteninvolves upper abdominal discomfort accompanied by frequent belching;gastric dyspepsia, which originates in the stomach; and intestinaldyspepsia, which originates in the intestines. It is understood thatthese and other mildly or acutely symptomatic forms of the condition areincluded in the definition of “dyspepsia” as used herein. In oneembodiment, the methods of the invention are used to prevent or reducethe severity of dyspepsia other than dyspepsia associated with gastricinflammation.

[0022] In another embodiment, the invention relates to treatinggastrointestinal disease. Inflammatory bowel disease (IBD) or irritablebowel syndrome (IBS) are gastrointestinal diseases which affect one-halfof all Americans during their lifetime, at a cost of greater than $2.6billion dollars for IBD and greater than $8 billion dollars for IBS. Thefrequency or severity of visceral hypersensitivity associated with IBD,IBS and other gastrointestinal diseases including inflammatorygastrointestinal diseases is exacerbated by stress. As disclosed herein,the methods of the invention can be useful for preventing or reducingthe severity of visceral hypersensitivity associated with astress-associated gastrointestinal disease such as, without limitation,ulcerative colitis (UC), Crohn's disease (CD), or irritable bowelsyndrome (IBS). Thus, the present invention provides a method ofpreventing or reducing the severity of visceral hypersensitivityassociated with a stress-associated gastrointestinal disease in asubject by systemically administering to the subject an effective amountof brimonidine or a pharmaceutically acceptable salt, ester, amide,sterioisomer or racemic mixture thereof.

[0023] The methods of the invention also can be useful for preventing orreducing the severity of tachycardia which is not associated withmyocardial ischemia. As used herein, the term “tachycardia” meansexcessive rapidity of heart rate and includes tachyarrhymthias. Inadults, the term tachycardia generally refers to a heart rate of greaterthan 100 beats per minute. The term tachycardia encompasses tachycardiassecondary to a variety of disorders other than myocardial ischemiaincluding, without limitation, paroxysmal tachycardia, in which thetachycardia is of sudden onset and cessation and either ventricular orsupraventricular, and nonparoxysmal tachycardia, which is a tachycardiaof slow onset, generally with a heart rate of 70 to 130 beats perminute. In one embodiment, a method of the invention prevents or reducesthe severity of an automatic tachycardia which is not associated withmyocardial ischemia. In another embodiment, a method of the inventionprevents or reduces the severity of tachycardia in an adult subject. Ina further embodiment, a method of the invention prevents or reduces theseverity of tachycardia in a subject who is a child.

[0024] Tachycardias to be treated according to a method of the inventioninclude those originating from any part of the heart such as ventriculartachycardias and supraventricular tachycardias, which can be classified,for example, into atrial and junctional (nodal) tachycardias. Thus, themethods of the invention can be useful for preventing or reducing theseverity of, for example, ventricular tachycardias, which are abnormallyrapid ventricular rhythms with aberrant ventricular excitation, often inexcess of 150 beats per minutes, generated within the ventricle andsometimes occurring in conjunction with atrioventricular dissociation.The methods of the invention further can be useful for preventing orreducing the severity of supraventricular tachycardias (SVT), which areregular tachycardias in which the point of stimulation is located abovethe bundle branches such as in the sinus node, atria or atrioventricularjunction or which arise from a large reentrant circuit including bothatrial and ventricular sites. In one embodiment, a method of theinvention is used to prevent or reduce the severity of an atrialtachycardia, which is characterized by a rapid cardiac rate generallybetween 160 and 190 beats per minutes and which originates from anatrial locus; such tachycardias include, but are not limited to,paroxysmal atrial tachycardias. In another embodiment, a method of theinvention is used to prevent or reduce the severity of a junctionaltachycardia, which is a tachycardia arising in response to impulsesoriginating in the atrioventricular junction and which is generallycharacterized by a heart rate greater than 75 beats per minute.Junctional tachycardias include nonparoxysmal and paroxysmal junctionaltachycardias, such as junctional tachycardias resulting from reentry orenhanced automaticity. It is understood that the methods also can beused to prevent or reduce the severity of, without limitation, doubletachycardias, in which two types of ectopic tachycardia are involved;sinus tachycardias, which originate in the sinus node and can beassociated with shock, hypotension, congestive heart failure or fever;orthostatic tachycardia, which is characterized by a disproportionaterapidity of heart rate upon rising from a reclining to a standingposition; and chaotic atrial tachycardia, which is characterized byatrial rates of 100 to 130 beats per minute, markedly variable P wavemorphology and irregular P—P intervals (Anderson, supra, 1994).

[0025] Tachycardias to be treated according to a method of the inventioncan be associated with one or more disorders such as pulmonary disease,diabetes, or surgical trauma and can occur, for example, in the elderly.As an example, chaotic atrial tachycardia (multifocal atrialtachycardia) can be present, for example, in patients with chronicobstructive pulmonary disease, in patients with diabetes, and in theelderly. As a further example, nonparoxysmal junctional tachycardia canbe associated, for example, with surgical trauma. It is understood thatthese and a variety of well known automatic and other tachycardias whichare not associated with myocardial ischemia can be prevented or reducedin severity according to the methods of the invention. In anotherembodiment, the invention provides a method of preventing or reducingthe severity of tachycardias of all types including those associatedwith myocardial ischemia.

[0026] The methods of the invention also can be useful for preventing orreducing the severity of panic attack, a common disorder with aprevalence of around 3% in the general population (Potokar and Nutt,Int. J. Clin. Pract. 54: 110-114 (2000)). Panic disorder involvingrecurrent panic attacks is typically observed in young adults, with anaverage age of onset of 24 years, and is more common in females than inmales. The term “panic attack,” as used herein, means a discrete periodof intense fear or discomfort accompanied by one or more of thefollowing symptoms: accelerated heart rate or palpitation; chest pain;chills or hot flushes; derealization or depersonalization; fear ofdying; fear of losing control or going crazy; dizziness or faintness;feelings of choking; nausea or abdominal distress; paraesthesia;sensations of shortness of breath or smothering; sweating; or tremblingor shaking. A panic attack typically begins with the sudden onset ofintense apprehension or fear and generally has a duration of about 5 to20 minutes. The term panic attack encompasses both full-blown andlimited-symptom attacks; full-blown attacks involve four or more of theabove symptoms while limited-symptom attacks involve fewer than foursymptoms. A method of the invention can entirely prevent a panic attack,or can prevent or reduce the severity of one or any combination of theattendant symptoms described above.

[0027] Some patients with panic attacks develop “panic disorder,” whichalso can be prevented or reduced in severity using brimonidine accordingto a method of the invention. The term panic attack, as used herein,encompasses panic disorder, which is defined as recurrent panic attacksin conjunction with persistent concern over additional episodes or theconsequences of the attacks or a notable change in behavior experiencedfor at least one month following one or more panic attacks. Panicdisorder can be associated with other psychiatric conditions such asdepression.

[0028] The central sympathetic nervous system can play a critical rolein the development of insulin-resistance and hypertension whichcharacterize type II diabetes (Rocchini et al., Hypertension 33[partII]:548-553 (1999)). Further provided herein is a method of preventingor reducing the severity of type II diabetes, a disorder characterizedby hypertension, hyperlipidemia and insulin-resistance and which isexacerbated by stress. As disclosed herein, brimonidine or apharmaceutically acceptable salt, ester, amide, sterioisomer or racemicmixture thereof, can be systemically administered to a subject in orderto prevent or reduce the severity of type II diabetes in the subject.

[0029] The methods of the invention also can be useful for preventing orreducing the severity of a non-inflammatory dermatological condition.Such methods can be useful, for example, for preventing or reducing theseverity of one or more symptoms such as itching or other discomfortassociated with a non-inflammatory dermatological condition. As usedherein, the term “non-inflammatory dermatological condition” means anydermatosis or other skin disease or condition that is not caused oraccompanied by inflammation. A non-inflammatory dermatological conditionto be treated according to a method of the invention can originate or beexacerbated under stressful conditions. Non-inflammatory dermatologicalconditions encompass, without limitation, non-inflammatory dermatosesincluding non-inflammatory blistering diseases such as epidermolysisbullosa and porphyria; ichthyosis; keratosis pilaris; juvenile plantardermatosis (JPD); lichen plantus dermatosis; and xerosis. One skilled inthe art understands that these and other non-inflammatory dermatologicalconditions known in the art can be treated by a method disclosed herein.

[0030] In a separate embodiment, the invention provides a method ofpreventing or reducing the severity of a stress-associated inflammatorydermatological condition in a subject by systemically administering tothe subject an effective amount of brimonidine or a pharmaceuticallyacceptable salt, ester, amide, sterioisomer or racemic mixture thereof.Such methods can be useful, for example, in preventing or reducing theseverity of one or more symptoms such as itching or other discomfortassociated with the inflammatory dermatological condition. Any of avariety of inflammatory dermatological conditions are encompassed by themethods of the invention including, without limitation, any of a varietyof forms of acute or chronic dermatitis such as psoriasis, allergicdermatitis such as allergic contact dermatitis, atopic dermatitis,dermatitis calorica, contact dermatitis, cosmetic dermatitis, eczema,exfoliative dermatitis, factitial dermatitis, irritant dermatitis,lichen simplex chronicus, marine dermatitis, neurodermatitis, perioraldermatitis, phototoxic dermatitis, seborrheic dermatitis, stasisdermatitis and dermatitis vegetans.

[0031] The methods of the invention can be useful for preventing orreducing the severity of a variety of disorders of muscle contraction,which are conditions that result, at least in part, from inappropriatemuscle contraction. Disorders of muscle contraction to be treatedaccording to a method of the invention include, without limitation,disorders of skeletal muscle contraction, disorders of smooth musclecontraction, disorders of muscle contraction associated with a gland,and disorders of cardiac muscle contraction such as congestive heartfailure; these and other disorders to be prevented or reduced inseverity according to a method of the invention include those in whichthe myocytes are innervated as well as those in which the myocytes arenot innervated. As non-limiting examples, the methods of the inventioncan be useful for preventing or reducing the severity of a disorder ofmuscle contraction such as back or other muscle spasm; musclecontraction associated with cystitis; muscle contraction associated withnon-bacterial prostatitis; muscle contraction associated with teethgrinding; muscle contraction associated with tension type headache; andmuscle contraction associated with congestive heart failure.

[0032] The methods of the invention can be useful, for example, forpreventing or reducing the severity of a muscle spasm such as a backspasm. Muscle spasms are well known in the art. As used herein, the term“spasm” means a sudden, involuntary contraction of a muscle or a groupof muscles, accompanied by pain and interference with function. A spasmcan produce, for example, involuntary movement or distortion. In oneembodiment, a method of the invention prevents or reduces the severityof a back spasm.

[0033] In one embodiment, a method of the invention is useful forpreventing or reducing the severity of muscle contraction associatedwith cystitis. As used herein, the term “cystitis” means inflammation ofthe urinary bladder. The term cystitis encompasses, yet is not limitedto, allergic cystitis, bacterial cystitis, acute catarrhal cystitis,cystic cystitis, diphtheritic (croupous) cystitis, eosinophiliccystitis, exfoliative cystitis, cystitis follicularis, cystitisglandularis, incrusted cystitis, chronic interstitial (panmural,submucous) cystitis, mechanical cystitis, cystitis papillomatosa andcystitis senilis feminarum. See, for example, Anderson, supra, 1994.Cystitis can be accompanied by one or more of the following clinicalsymptoms: frequent urination, burning on urination, suprapubicdiscomfort, lassitude, cloudy or blood-tinged urine and sometimeslow-grade fever (Bennett and Plum (Eds.), Cecil Textbook of MedicineSixth Edition, W. B. Saunders Company, Philadelphia 1996). One skilledin the art understands that the muscle contraction associated with anyof these or other forms of mild, severe, acute or chronic cystitis canbe treated according to a method of the invention.

[0034] As disclosed herein, a method of the invention also can be usefulfor preventing or reducing the severity of muscle contraction associatedwith non-bacterial prostatitis. Symptoms of prostatic inflammation areexperienced by about 50% of men in adult life; of these, about 95%result from factors other than bacterial infection. As used herein, theterm “non-bacterial prostatitis” is synonymous with “abacterialprostatitis” and means inflammation of the prostate not resulting frombacterial infection. Non-bacterial prostatitis encompasses, yet is notlimited to, chronic non-bacterial prostatitis, allergic or eosinophilicprostatitis and non-specific granulomatous prostatitis. It is understoodthat the term non-bacterial prostatitis includes, without limitation,prostatitis of unknown etiology characterized by abnormal expressedprostatic secretions (EPS) and normal bacterial cultures. In some cases,non-bacterial prostatitis can be effectively treated with antibiotics orstress management (Bennett and Plum, supra, 1996). It is understood thatmuscle contraction associated with these or other forms of mild, severe,acute or chronic non-bacterial prostatitis can be treated according to amethod of the invention.

[0035] In another embodiment, a method of the invention is useful forpreventing or reducing the severity of muscle contraction associatedwith tension type headache (TTH), which is a common form of headacheaffecting as many as 90% of adult Americans. As used herein, the term“tension type headache” means a headache caused, at least in part, bymuscle contraction, which may be triggered, for example, by stress orexertion. The term “tension type headache” encompasses episodic andchronic headache and includes but is not limited to common tensionheadaches. Tension type headaches generally involve the posterior of thehead and neck, although they may also appear at the top or front of theskull and are further generally characterized by symmetry and anon-disabling severity. Although not all may be present, diagnosticfeatures of tension type headache include bilateral pain; mild tomoderate severity; pressing-like character with a stable profile;accentuation as the day progresses; possible high frequency such asdaily or continuously; and relative rarity of migrainous features suchas nausea, photosensitivity, phonosensitivity and aggravation byphysical activity such as head movement.

[0036] Tension type headaches result from tightening of muscles of theface, neck and scalp due, for example, to stress, overwork, eyestrain orpoor posture. Such headaches can last for days or weeks and can causepain of varying intensity. Tension type headaches occurring over anextended period of time such as several weeks or months are denotedchronic tension headaches and are encompassed by the term tension typeheadache as used herein.

[0037] Tension type headaches can be distinguished from migraines by theabsence of vascular features and symptoms such as nausea, vomiting andsensitivity to light and the absence of an aura (Spira, Austr. FamilyPhys. 27: 597-599 (1998). The term tension type headache, which refersto headaches without a significant vascular component, is used incontradistinction to tension-vascular headaches, cluster headaches,migrainous headaches and other headaches with a major vascularcomponent. However, the methods of the invention also can be useful forpreventing or reducing the severity of sensory hypersensitivityassociated with other headaches including, but not limited to,cervicogenic headache, post-traumatic headache, cluster headache andtemporomandibular joint disorder (TMJ).

[0038] The methods of the invention also can be useful for preventing orreducing the severity of sensory hypersensitivity associated withmigraine, a headache that plagues more than 10% of the population andthat may be associated with a vascular component. In one embodiment, themethods of the invention prevent or reduce the severity of an ocularhypersensitivity associated with migraine, for example, photophobia. Themethods of the invention are useful for preventing or reducing theseverity of sensory hypersensitivity associated with any of a variety offorms of migraine including, but not limited to, migraine without aura(“MO”), migraine with aura (“MA”), and migrainous disorder. Sensoryhypersensitivity to be prevented or reduced in severity according to amethod of the invention further can be associated with, for example,abdominal migraine, acute confusional migraine, basilar (basilar artery)migraine, hemiplegic or familial hemiplegic migraine, fulguratingmigraine, ocular (ophthalmic) migraine, ophthalmoplegic migraine orretinal migraine. In addition, the methods of the invention can beuseful for preventing or reducing the severity of sensoryhypersensitivity associated with a migraine equivalent, in which thereis a migraine aura without headache. Migraine auras are the abnormalvisual, motor, psychic, paresthesic or other neurologic abnormalitiesthat accompany a migraine. See Elrington, J. Neurol. Neurosura.Psychiatry 72 Supple. II:ii10-ii15 (2002); Anderson, supra, 1994;Bennett and Plum, supra, 1996.

[0039] The methods of the invention can be useful for preventing orreducing the severity of one or more of a variety of types of sensoryhypersensitivity associated with migraine. Such sensory hypersensitivityincludes, but is not limited to, nausea; vomiting; diarrhea; photophobia(light intolerance); and phonophobia (noise intolerance). Such sensoryhypersensitivity also includes visual abnormalities such as brightflashing lights (scintillation or fortification scotomata) or amonocular (retinal) visual abnormality or hemianoptic loss of vision;paresthesia (abnormal touch sensation) such as unilateral paresthesia;aphasia (loss of speech or comprehension); hemiparesis (muscularweakness or incomplete paralysis on one side of the body); hemisensorydefect; or vertigo, ataxia (loss of muscular coordination) or diplopia.It is understood that the methods of the invention can be useful forpreventing or reducing the severity of one of these or other types ofsensory hypersensitivity occurring prior to, during, or subsequent tomigraine headache, or occurring in the absence of headache as part of amigraine equivalent.

[0040] The methods of the invention also can be useful for preventing orreducing the severity of one or more of a variety of types of sensoryhypersensitivity associated with other disorders such as fibromyalgia,also known as fibrositis. Fibromyalgia is a disorder involving chronic,widespread musculoskeletal pain and tenderness at multiple sites in theabsence of signs of connective tissue or other musculoskeletal disease.In particular, fibromyalgia is defined by pain or tenderness at 11 of 18or more sites as defined by the American College of Rheumatology.Fibromyalgia frequently is associated with disturbed sleep, chronicfatigue, headaches and irritable bowel syndrome (Bennett and Plum,supra, 1996).

[0041] A variety of types of sensory hypersensitivity can be associatedwith fibromyalgia and can be prevented or reduced in severity accordingto a method of the invention, including, without limitation,hypersensitivity to light, noise, touch or odors, cold or heatintolerance, nausea or allergic-like symptoms such as rhinitis, itching,or rash in the absence of a true allergy. One skilled in the artunderstands that the methods of the invention can be useful forpreventing or reducing the severity of any of these or other types ofsensory hypersensitivity associated with fibromyalgia.

[0042] The methods of the invention further can be useful for preventingor reducing the severity of a stress-associated behavioral disorder,which is any behavioral disorder which is induced or exacerbated bystress. As non-limiting examples, a stress-associated behavioraldisorder can be a compulsive or repetitive detrimental behavior which isinduced or exacerbated by stress such as, without limitation,over-eating or obesity, obsessive compulsive disorder (OCD), tics,Tourette syndrome (TS), alcohol use, drug use, gambling, self-inflictedinjurious behavior such as scratching or hair-pulling, or sexualimpotency or arousal. In one embodiment, the stress-associatedbehavioral disorder is a disorder other than drug use. In anotherembodiment, the stress-associated behavioral disorder is a disorderother than drug or alcohol use.

[0043] The methods of the invention further can be useful for preventingor reducing the severity of a stress-associated psychiatric disorder,which is any psychiatric disorder which is induced or exacerbated bystress. As a non-limiting example, the methods of the invention can beused to prevent or reduce the severity of a psychiatric disorder such asschizophrenia.

[0044] Also provided herein is a method of preventing or reducing theseverity of an ocular condition in a subject by systemicallyadministering to the subject an effective amount of brimonidine or apharmaceutically acceptable salt, ester, amide, sterioisomer or racemicmixture thereof. As disclosed herein, brimonidine can act as aneuroprotective agent, preventing, for example, retinal damage in anumber of ocular conditions affecting the neurosensory retina. Ocularconditions which can be prevented or reduced in severity usingbrimonidine according to a method of the invention include, withoutlimitation, diabetic retinopathy; macular edema such as macular edemaassociated with diabetes mellitus or other conditions; retinaldegeneration such as age-related macular degeneration or retinitispigmentosa; inflammatory disorders of the retina; vascular occlusiveconditions of the retina such as retinal vein occlusions or branch orcentral retinal artery occlusions; retinopathy of prematurity;retinopathy associated with blood disorders such as sickle cell anemia;damage following retinal detachment; damage or insult due to vitrectomysurgery or retinal surgery; and other retinal damage includingtherapeutic damage such as that resulting from laser treatment of theretina, for example, pan-retinal photocoagulation for diabeticretinopathy or photodynamic therapy of the retina, for example, forage-related macular degeneration as well as other ocular conditions suchas ocular itch. Ocular conditions that can be prevented or reduced inseverity according to a method of the invention further include, withoutlimitation, genetic and acquired optic neuropathies such as opticneuropathies characterized primarily by loss of central vision, forexample, Leber's hereditary optic neuropathy (LHON), autosomal dominantoptic atrophy (Kjer disease) and other optic neuropathies such as thoseinvolving mitochondrial defects, aberrant dynamin-related proteins orinappropriate apoptosis. See, for example, Carelli et al., Neurochem.Intl. 40:573-584 (2002); and Olichon et al., J. Biol. Chem.278:7743-7746 (2003).

[0045] The methods of the invention can be useful for preventing orreducing the severity of a stress-associated condition withoutconcomitant sedation. Sedation, as used herein, is a term that means areduction in motor activity. The phrase “without concomitant sedation,”as used herein, means that relatively little reduction in motor activityaccompanies the reduction in severity of one or more symptoms of astress-associated condition at one or more doses of drug. A druggenerally acts “without concomitant sedation” if, upon peripheraladministration, the dose required to produce a 20% reduction in motoractivity is at least 3-fold greater than the dose required to produce asignificant reduction in one or more symptoms of the stress-associatedcondition. As shown in FIG. 6, brimonidine but not tizanidine orclonidine could be administered at doses that produced a reduction inthe sensitization score (solid line, left axis) with less than a 20%increase in sedation (broken line, right axis). As non-limitingexamples, the dose required to produce a 20% reduction in motor activitycan be at least 4-fold greater than, 5-fold greater than, 6-fold greaterthan, 7-fold greater than, 8-fold greater than, 9-fold greater than,10-fold greater than, 25-fold greater than, 50-fold greater than,100-fold greater than, 200-fold greater than, 500-fold greater than,1000-fold greater than, 2000-fold greater than, or 5000-fold greaterthan the dose required to produce a significant reduction in one or moresymptoms of a stress-associated condition. Methods of determining theextent of a reduction in severity of symptoms of a stress-associatedcondition and the extent of sedation are well known in the art.

[0046] The term “brimonidine,” as used herein, means a compound havingthe formula

[0047] or a pharmaceutically acceptable derivative thereof such as asalt, ester, amide, sterioisomer, racemic mixture, polymorph, hydrate orsolvate. Such a pharmaceutically acceptable derivative can havesubstantially the activity of5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline D-tartrate (1:1) inreducing tactile hypersensitivity without concomitant sedation insulprostone-treated mice. The term brimonidine encompasses, withoutlimitation, Alphagan™ and UK14304. Brimonidine, and pharmaceuticallyacceptable salts, esters, amides, sterioisomers and racemic mixturesthereof, is commercially available, for example, as Alphagan™(Allergan). In addition, brimonidine and pharmaceutically acceptablesalts, esters, amides, sterioisomers and racemic mixtures thereof can beprepared by routine methods as described below in Example I. See, also,U.S. Pat. No. 6,323,204.

[0048] Thus, it is understood that the methods of the inventionencompass the use of pharmaceutically acceptable salts, esters andamides derived from the formula representing brimonidine. Suitablepharmaceutically acceptable salts of brimonidine include, withoutlimitation, acid addition salts, which can be formed, for example, bymixing a solution of brimonidine with a solution of an appropriate acidsuch as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid,succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid,carbonic acid or phosphoric acid. Pharmaceutically acceptable saltsfurther include, yet are not limited to, acid phosphate, acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide,hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate,malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium,oleate, oxalate, pamoate (embonate), palmitate, pantothenate,phosphate/diphosphate, polygalacturonate, saccharate, salicylate,stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate,p-toluene sulphonate salts, tosylate, triethiodide and valerate. In oneembodiment, a method of the invention is practiced with brimonidinetartrate.

[0049] It further is understood that functional groups of brimonidinecan be modified, for example, to enhance the pharmacological utility ofthe compound. Such modifications, which are well within the knowledge ofthe skilled chemist and include, without limitation, esters, amides,ethers, N-oxides, and pro-drugs of brimonidine, are encompassed withinthe term “brimonidine” as used herein. Examples of modifications thatcan enhance activity include, for example, esterification such as theformation of C₁ to C₆ alkyl esters, preferably C₁ to C₄ alkyl esters,wherein the alkyl group is a straight or branched chain. Otheracceptable esters include, for example, C₅ to C₇ cycloalkyl esters andarylalkyl esters such as benzyl esters. Such esters can be prepared fromthe compounds described herein using conventional methods well known inthe art of organic chemistry.

[0050] Other pharmaceutically acceptable modifications include theformation of amides. Useful amide modifications include, for example,those derived from ammonia; primary C₁ to C₆ dialkyl amines, where thealkyl groups are straight or branched chain; and arylamines havingvarious substitutions. In the case of secondary amines, the amine alsocan be in the form of a 5 or 6 membered ring. Methods for preparingthese and other amides are well known in the art.

[0051] It is further understood that chemically distinct enantiomers andtautomers of brimonidine are encompassed within the term “brimonidine”and can be useful in the methods of the invention. Furthermore, incrystalline form, a compound may exist as polymorphs; in the presence ofa solvent, a compound may form a solvate, for example, with water or acommon organic solvent. Such polymorphs, hydrates and other solvates ofbrimonidine also are encompassed within the term “brimonidine” and canbe useful in the methods of the invention disclosed herein.

[0052] It is understood that pharmaceutical compositions containingbrimonidine can be useful in the methods of the invention. Such apharmaceutical composition includes brimonidine and optionally includesan excipient such as a pharmaceutically acceptable carrier or a diluent,which is any carrier or diluent that has substantially no long term orpermanent detrimental effect when administered to a subject. Anexcipient generally is mixed with active compound, or permitted todilute or enclose the active compound. A carrier can be a solid,semi-solid, or liquid agent that acts as an excipient or vehicle for theactive compound. Examples of solid carriers include, without limitation,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, polyalkylene glycols, talcum, cellulose, glucose,sucrose and magnesium carbonate. Suppository formulations can include,for example, propylene glycol as a carrier. Examples of pharmaceuticallyacceptable carriers and diluents include, without limitation, water,such as distilled or deionized water; saline; aqueous dextrose,glycerol, ethanol and the like. It is understood that the activeingredients can be soluble or can be delivered as a suspension in thedesired carrier or diluent.

[0053] A pharmaceutical composition also can optionally include one ormore agents such as, without limitation, emulsifying agents, wettingagents, sweetening or flavoring agents, tonicity adjusters,preservatives, buffers or anti-oxidants. Tonicity adjustors useful in apharmaceutical composition include, but are not limited to, salts suchas sodium acetate, sodium chloride, potassium chloride, mannitol orglycerin and other pharmaceutically acceptable tonicity adjustors.Preservatives useful in pharmaceutical compositions include, withoutlimitation, benzalkonium chloride, chlorobutanol, thimerosal,phenylmercuric acetate, and phenylmercuric nitrate. Various buffers andmeans for adjusting pH can be used to prepare a pharmaceuticalcomposition, including, but not limited to, acetate buffers, citratebuffers, phosphate buffers and borate buffers. Similarly, anti-oxidantsuseful in pharmaceutical compositions are well known in the art andinclude, for example, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition useful inthe methods of the invention. See, for example, Remington'sPharmaceutical Sciences Mack Publishing Company, Easton, Pa. 16^(th)Edition 1980. Furthermore, a composition containing brimonidine may beadministered in conjunction with one or more other therapeuticsubstances, in the same or different pharmaceutical composition and bythe same or different routes of administration.

[0054] Brimonidine, or a pharmaceutically acceptable salt, ester, amide,sterioisomer or racemic mixture thereof, is administered in an effectiveamount. Such an effective amount generally is the minimum dose necessaryto achieve the desired prevention or reduction in severity of one ormore symptoms of a stress-associated condition, for example, that amountroughly necessary to reduce the discomfort caused by thestress-associated condition to tolerable levels. Such a dose generallyis in the range of 0.1-1000 mg/day and can be, for example, in the rangeof 0.1-500 mg/day, 0.5-500 mg/day, 0.5-100 mg/day, 0.5-50 mg/day, 0.5-20mg/day, 0.5-10 mg/day or 0.5-5 mg/day, with the actual amount to beadministered determined by a physician taking into account the relevantcircumstances including the severity and type of stress-associatedcondition, the age and weight of the patient, the patient's generalphysical condition, and the pharmaceutical formulation and route ofadministration. Suppositories and extended release formulations also canbe useful in the methods of the invention, including, for example,dermal patches, formulations for deposit on or under the skin andformulations for intramuscular injection.

[0055] A pharmaceutical composition useful in the methods of theinvention can be administered to a subject by a variety of meansdepending, for example, on the type of condition to be treated, thepharmaceutical formulation, and the history, risk factors and symptomsof the subject. Routes of administration suitable for the methods of theinvention include both systemic and local administration. Asnon-limiting examples, a pharmaceutical composition useful forpreventing or reducing the severity of a stress-associated condition canbe administered orally; parenterally; by subcutaneous pump; by dermalpatch; by intravenous, intra-articular, subcutaneous or intramuscularinjection; by topical drops, creams, gels or ointments; as an implantedor injected extended release formulation; by subcutaneous minipump orother implanted device; by intrathecal pump or injection; or by epiduralinjection. Depending on the mode of administration, brimonidine can beincorporated in any pharmaceutically acceptable dosage form such as,without limitation, a tablet, pill, capsule, suppository, powder,liquid, suspension, emulsion, aerosol or the like, and can optionally bepackaged in unit dosage form suitable for single administration ofprecise dosages, or sustained release dosage forms for continuouscontrolled administration.

[0056] A method of the invention can be practiced by peripheraladministration of brimonidine, or a pharmaceutically acceptable salt,ester, amide, sterioisomer or racemic mixture thereof. As used herein,the term “peripheral administration” or “administered peripherally”means introducing brimonidine, or a pharmaceutically acceptable salt,ester, amide, sterioisomer or racemic mixture thereof, into a subjectoutside of the central nervous system. Peripheral administrationencompasses any route of administration other than direct administrationto the spine or brain.

[0057] Peripheral administration can be local or systemic. Localadministration results in significantly more of a pharmaceuticalcomposition being delivered to and about the site of localadministration than to regions distal to the site of administration.Systemic administration results in delivery of a pharmaceuticalcomposition essentially throughout at least the entire peripheral systemof the subject.

[0058] Routes of peripheral administration useful in the methods of theinvention encompass, without limitation, oral administration, topicaladministration, intravenous or other injection, and implanted minipumpsor other extended release devices or formulations. A pharmaceuticalcomposition useful in the invention can be peripherally administered,for example, orally in any acceptable form such as in a tablet, liquid,capsule, powder, or the like; by intravenous, intraperitoneal,intramuscular, subcutaneous or parenteral injection; by transdermaldiffusion or electrophoresis; topically in any acceptable form such asin drops, creams, gels or ointments; and by minipump or other implantedextended release device or formulation.

[0059] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I Preparation of Brimonidine

[0060] This example describes preparation of brimonidine(5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline).

[0061] Preparation 6-amino-5-bromoquinoxaline Hydrobromide

[0062] 6-Aminoquinoxaline (2.08 g, 14.4 mmol) was dissolved in 11.5 mlglacial acetic acid. The solution was cooled in water while a solutionof bromine (0.74 ml, 2.3 g, 14.4 mmol) in 1.5 ml glacial acetic acid wasadded slowly over 15 minutes. After stirring for an additional 30minutes, the orange red solid formed was filtered off and washedthoroughly with dry ether. The solid was dried in vacuo overnight toyield 4.44 g crude product (a yield of 100%). The compound,6-amino-5-bromoquinoxaline hydrobromide, had no definite melting point.A phase change from fine powder to red crystals was observed at about2200 C. Decomposition was observed at about 2450 C. The material wasused directly for preparation of 6-amino-5-bromoquinoxaline as follows.

[0063] 6-Amino-5-Bromoquinoxaline

[0064] Crude 6-amino-5-bromoquinoxaline from above was dissolved inwater, and saturated sodium bisulfite solution was added until theresulting solution tested negative with starch-iodide paper. Thesolution was then basified with 2N sodium hydroxide and extractedthroroughly-with ethyl acetate. The organic extract was dried overmagnesium sulfate and concentrated under reduced pressure to give thefree base. The crude product was recrystallized from boiling benzene togive yellow crystals, m.p. 155-6° C. Using various analyticalprocedures, the yellow crystals were determined to be6-amino-5-bromoquinoxaline. The yield was 82%.

[0065] 6-Bromo-6-isothiocyanatoquinoxaline

[0066] The crude hydrobromide product described above (4.27 g, 14.0mmol) was dissolved in 60 ml of water; thiophosgene (Aldrich, 1.28 ml,16.8 mmol) was added in small portions with vigorous stirring. After 2hours, the red color of the solution was discharged. The solid formedwas filtered off and washed thoroughly with water. After drying in vacuoat 25° C., 3.38 g of brick red crystals were obtained, m.p. 157-8° C.,representing a yield of 90%. A portion of this material was furtherpurified by column chromatography to give white crystals, m.p. 157-80 C.Using various analytical procedures, these crystals were determined tobe 5-bromo-6-isothiocyanatoquinoxaline.

[0067] 5-Bromo-6(—N-(2aminoethyl)thioureido)quinoxaline

[0068] A solution of the isothiocyanate (3.25 g, 12.2 mmol) in 145 mlbenzene was added to a solution of ethylenediamine (Aldrich, 5.43 g,90.0 mmol) in 18 ml benzene at 25° C. over 2 hours. After stirring for afurther 30 minutes, the supernatant was poured off. The oil whichremained was washed by swirling with dry ether three times and useddirectly for the next step.

[0069] A portion of this product was further purified by columnchromatography (SiO₂, CHCl₃) for characterization. A white solid wasrecovered which decomposed at 175° C. with gas evolution (puffing). Thiswhite solid was determined to be 5-bromo-6(—N-2-(aminoethyl)thioureido)quinoxaline.

[0070] 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline

[0071] The crude product from above was dissolved in 100 ml dry methanoland the brown solution was refluxed for 19 hours until hydrogen sulfidegas was no longer evolved. The mixture was cooled to room temperatureand concentrated to about 50 ml. The yellow solid was filtered off anddried in vacuo; the solid weighed 2.52 g (a yield of 70%) and had amelting point of 242-4° C.

[0072] As the crude product was insoluble in most common organicsolvents, initial purification was achieved by an acid-base extractionprocedure. Crude product (23 g) was dissolved in 100 ml 0.5Nhydrochloric acid. The turbid yellow solution was filtered to give aclear orange yellow solution which was extracted twice with ethylacetate (10 ml each extraction). The aqueous phase was cooled to 0° C.and basified with 6N sodium hydroxide, keeping the temperature of thesolution below 15° C. at all times. The yellow solid which precipitatedwas filtered off and washed thoroughly with water until the washingswere neutral to pH paper. The solid was dried overnight in vacuo to give1.97 g yellow solid, m.p. 249-250° C. The recovery was about 88%.

[0073] Further purification was achieved by recrystallization. Thepartially purified product from above was dissolved inN,N-dimethylformamide (about 17 ml/g) at 100° C. with vigorous stirring.The solution was filtered hot and set aside to cool overnight. Thebright yellow crystals were collected by filtration, m.p. 252-253° C.Recovery was from 65-77%. Using various analytical procedures, thebright yellow solid was determined to be5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline.

EXAMPLE II Mouse Models with Different Mechanisms of SensorySensitization

[0074] This example demonstrates that the increased sympathetic tone ofα-2A and α-2C knockout mice enhances induction of tactilehypersensitivity by α-1 receptor activation.

[0075] A. Sulprostone-Induced Tactile Hypersensitivity is Driven by theSympathetic Nervous System While Phenylephrine-Induced TactileHypersensitivity is Independent of Sympathetic Nervous System Input

[0076] To dissect the contribution of the sympathetic nervous system tosensory sensitization, mouse models having different mechanisms ofsensory sensitization were developed. Tactile hypersensitivity wasmeasured in mice following intrathecal or intraperitoneal injection ofan inducing agent by scoring the response to light stroking of the mouseflank with a paintbrush. To mimic increased sympathetic tone,phenylephrine, an α-1 adrenergic receptor agonist, was injected. Asshown in FIGS. 1a and 1 b, intrathecal (i.t.) or intraperitoneal (i.p.)dosing of phenylephrine caused tactile hypersensitivity, withsignificant responses observed starting at doses of 3 ng i.t. and 3ng/kg i.p. Induction of tactile hypersensitivity was α-1 receptordependent, as evidenced by the ability of the α-1 receptor antagonist5-methyl urapidil (5-MU) to block the hypersensitive response wheninjected intraperitoneally.

[0077] The activity of a synthetic EP₁/EP₃ receptor-selectiveprostaglandin agonist, sulprostone, also was assayed. As shown in FIG.1c, increasing doses of intrathecal sulprostone elicited dose-dependenttactile hypersensitivity; doses of 100 and 200 ng caused a significanthypersensitive response. Coadministration of a specific EP₁ receptorantagonist completely blocked the sulprostone-induced tactilehypersensitivity, demonstrating that sulprostone mediates tactilehypersensitivity through activation of the EP₁ receptor.

[0078] In a third mouse model, chemical sensitization was induced byinjection of increasing intrathecal doses of NMDA, which may activateNMDA channels on post-synaptic dorsal horn neurons (Woolf et al.,Science 288:1765-1769 (2000)). Intrathecal NMDA resulted in adose-dependent tactile hypersensitivity with a maximal effect at a 100ng dose. The hypersensitivity was blocked with the NMDA antagonist,memantine, as shown in FIG. 1d.

[0079] To assess whether the three stimuli sensitize sensory pathways bydifferent mechanisms, a set of pharmacological agents was assayed forthe ability to prevent or ameliorate tactile hypersensitivity. As shownin Table 1, each receptor antagonist (5-MU, the EP₁ receptor antagonistor memantine) blocked only tactile hypersensitivity resulting from thecorresponding receptor agonist (phenylephrine, sulprostone or NMDA,respectively). Gabapentin, which is used clinically to alleviateneuropathic pain by reducing spinal sensitization, also was assayed forthe ability to block tactile hypersensitivity. Gabapentin inhibitedtactile hypersensitivity elicited by sulprostone and NMDA, but not byphenylephrine, further demonstrating differences between the sensorypathways involved by different stimuli. TABLE 1 Receptor antagonists andclinically used analgesics inhibit chemically-induced mechanicalhypersensitivity EP₁ Vehicle 5-MU antagonist Memantine GabapentinPhenylephrine 14.3 ± 0.7**  5.0 ± 1.0  9.8 ± 0.7** 11.0 ± 0.7** 13.0 (±0.6)** (100 ng/kg I.P.) Sulprostone 13.2 ± 0.8** 12.0 ± 1.0**  4.0 ± 1.214.3 ± 0.8**  3.2 ± 0.5 (200 ng IT) NMDA 14.2 ± 1.0** 13.3 ± 0.8** 11.4± 1.53*  4.2 ± 0.9  3.7 ± 0.8 (100 ng IT)

[0080] α-2 knockout mice were provided by Dr. Brian Kobilka (StanfordUniversity; Link et al., Mol. Pharmacol. 48:48-55 (1995); Altman et al.,Mol. Pharmacol. 56:154-161 (1999)). The α-2 knockout mice have a C57BL/6background and were bred from homozygous knockout mice breeding pairs.Age and sex matched C57BL/6 wildtype mice were used as controls.

[0081] Sulprostone (Cayman Chemical; Ann Arbor, Mich.) and NMDA (Sigma;St Louis, Mo.) were dissolved in dimethyl sulfoxide (DMSO). The EP₁receptor antagonist

[0082] synthesized essentially as described in U.S. Pat. No. 5,843,942,and gabapentin (Victor Medical; Irvine, Calif.) were dissolved in 50%DMSO, 50% saline. Memantine (1-amino-3,5-dimethyladamantanehydrochloride), an analog of the well known anti-viral agent amantadine(1-adamantanamine hydrochloride), was synthesized essentially asdescribed in U.S. Pat. No. 5,061,703 (see, also, Schneider et al., DtschMed. Wochenschr. 109:987 (1984)). 5-methylurapidil, brimonidine,phenylephrine, clonidine and guanethidine were obtained from Sigma anddissolved in saline. Prazosin (Sigma) and tizanidine (Biomol; PlymouthMeeting, Pa.) were dissolved in distilled water.

[0083] Spinal drug injections were performed as follows. Mice (20-30 g)were injected intrathecally as described in Hylden and Wilcox, Eur. J.Pharmacol. 67:313-316 (1980). Briefly, a sterile 30-gauge ½ inch needleattached to a microsyringe was inserted between the L5 and L6 vertebrae.The mouse was held firmly by the pelvic girdle in one hand, while thesyringe was held in the other hand at an angle of approximately 20°above the vertebral column. The needle was inserted into the tissue toone side of the L6 spinous process, into the groove between the spinousand transverse processes. The needle angle was decreased to about 10°,and the needle slowly advanced forward into the intervertebral spaceuntil a pop was felt and there was a visible serpentine tail movement.Compounds were slowly injected in the subarachnoid space in a volume of5 μl. Each compound was tested at multiple doses. The minimalefficacious dose was used for all subsequent experiments.

[0084] Sensitivity to light touch was quantified by scoring the responseof mice to light stroking of their flanks with a small paintbrush, whichis not normally painful. The mice were rated on the following scale onceevery 5 minutes between 15 and 50 minutes post injection: a score of “2”was given to animals showing aggressive escape responses along withsqueaking and biting at the brush; a score of “1” was given to animalsexhibiting mild squeaking with attempts to escape; and a score of “0”was given if the animal showed no response to the light stroking of thepaintbrush. The scores were summed to generate a cumulative score of 0to 16 as described in Minami et al., Pain 57:217-223 (1994). Statisticalcalculations of significance for in vivo studies were done using atwo-tailed Students t-test.

[0085] Guanethidine sympathectomies were performed essentially asfollows. Animals were injected intraperitoneally with 50 mg/kgguanethidine (Malmberg and Basbaum, Pain 76:215-222 (1998)) before beingassessed for baseline tactile sensitivity 24 hours later. Animals thatexhibited normal tactile sensitivity were assayed for sensitivity tochemical induction of tactile hypersensitivity. Mice recovered from thesympathectomy six to eight days later as demonstrated by a return topre-sympathectomy responsiveness.

[0086] B. Increased Sympathetic Tone of α-2A and α-2C Knockout MiceEnhances their Sensitivity to Induction of Tactile Hypersensitivity byα-1 Receptor Activation

[0087] To assess whether sympathetic tone can influence susceptibilityto sensory sensitization, the sensitivity of α-2A and α-2C knockout miceto chemical induction of tactile hypersensitivity was compared to thesensitivity of wildtype mice. The α-2A and α-2C knockout mice did notexhibit baseline tactile hypersensitivity when compared to wildtypecontrols. First, the concentration of phenylephrine that elicits tactilehypersensitivity was compared in the knockout and wildtype mice. Asshown in FIG. 2, there was a dramatic leftward shift in thephenylephrine dose response in both the α-2A and α-2C knockout mice.These results demonstrate that the ability of phenylephrine to causetactile hypersensitivity was enhanced in both α-2 knockout mouse lines,with a greater enhancement in the α-2C knockout mice. In particular,compared with a strongly tactile hypersensitivity-inducing dose of 30ng/kg phenylephrine in the wildtype line, 0.1 and 0.3 ng/kgphenylephrine resulted in maximal hypersensitivity in the α-2C and α-2Aknockout mice, respectively. As further evidenced in FIG. 2, the gradualbiphasic dose-response in the wildtype mice became a steeperdose-response in both lines of knockout mice.

[0088] Systemic administration of guanethidine results in a functionalsympathectomy by depleting noradrenaline from sympathetic terminals. Inorder to test if shifts in the phenylephrine dose response curves weredue to increased sympathetic tone in the α-2 knockout mice, α-2Aknockout mice were chemically sympathectomized by guanethidine treatment(50 mg/kg i.p.) and assayed for phenylephrine-induced sensitivity 24-30hours later. In guanethidine-treated α-2A mice, the increasedsensitivity to phenylephrine was partly ablated so that the doseresponse was similar to the biphasic dose response observed in wildtypemice (see FIG. 2). These results confirm that increased sympathetic toneenhances sensory sensitization in α-2A knockout mice.

[0089] C. The Sympathetic Nervous System Enhances Sulprostone-InducedTactile Hypersensitivity

[0090] Sulprostone was injected intrathecally at increasingconcentrations into wildtype and α-2 knockout mice in order to determinewhether the knockout mice were more sensitive to sensitization ofprimary afferents. As shown in FIG. 3, the dose response of sulprostonewas identical in the wildtype and α-2C knockout mice, but was shifted tothe left in the α-2A knockout mice. In particular, a 30 ng dose wasmaximally effective in the α-2A knockout mice compared to a partiallyhypersensitivity-inducing dose of 100 ng and a maximal dose of 200 ng inthe wild-type and α-2C knockout mice. A guanethidine (50 mg/kg i.p.)chemical sympathectomy decreased the sensitivity of the α-2A knockoutmice to sulprostone. As shown in FIG. 3, the dose response ofsulprostone-induced tactile hypersensitivity was shifted approximately10-fold to the right in the α-2A knockout mice treated withguanethidine. These results demonstrate that the sympathetic nervoussystem enhances sulprostone sensitization.

[0091] D. The Sympathetic Nervous System does not Contribute toNMDA-Induced Tactle Hypersensitivity

[0092] To assess whether α-2 knockout mice are more sensitive to dorsalhorn sensitization by NMDA, wildtype and α-2 knockout mice were injectedwith varying concentrations of NMDA. As shown in FIG. 4, α-2A and α-2Cknockout mice are not more sensitive to NMDA than wildtype mice. Theseresults indicate that the sympathetic nervous system does not appear tocontribute to NMDA-induced tactile hypersensitivity.

[0093] In sum, these results demonstrate that α-2 knockout mice exhibitelevated levels of sympathetic nerve activity and further indicate thatthese mice exhibit enhanced sensitization which is specific to the siteand mode of stimulation.

EXAMPLE III Comparison of Activity of α-2 Agonists Brimonidine andClonidine

[0094] This example demonstrates that α-adrenergic agonists differ intheir ability to alleviate sensory hypersensitivity that is enhanced bythe sympathetic nervous system.

[0095] A. Brimonidine, but not Clonidine, AlleviatesSympathetically-Enhanced Tactile Hypersensitivity

[0096] Spinally administered α-2 adrenergic agonists alleviateneuropathic pain through a spinal α-2A receptor. To determine if theincreased sympathetic activity in α-2 knockout mice alters the analgesicactivity of the α-2 agonists, several agonists were assayed foractivity. The α-2 agonists brimonidine and clonidine were first testedin the NMDA model in which sensitization is not influenced by the basalsympathetic tone of the knockout mice. Intrathecal co-administration ofNMDA with either clonidine or brimonidine resulted in completeinhibition of tactile hypersensitivity in the wildtype and α-2C (FIGS.5a and c, respectively) knockout mice. As expected, neither clonidinenor brimonidine inhibited NMDA-induced tactile hypersensitivity in theα-2A knockout mice (FIG. 5c), consistent with previous studies showingthat a spinal α-2A adrenergic receptor subtype mediates analgesicactions of α-2 adrenergic agonists (Lakhlani et al., Proc. Natl. Acad.Sci. USA 94:9950-9955 (1997); Stone et al., J. Neurosci. 17:7157-1765(1997); Hunter et al., Br. J. Pharmacol. 122:1339-1344 (1997)). The samepattern of analgesic activity of brimonidine also was observed in thesulprostone-induced tactile hypersensitivity model, which is sensitiveto sympathetic tone (see FIGS. 5b and d). In contrast, the resultsobtained with clonidine were strikingly different: clonidine wasanalgesic in wildtype mice, but not in α-2A or α-2C knockout mice(compare FIGS. 5b and d). These results demonstrate that α-2pan-agonists can have differential activity in sympathetically-enhancedconditions, with brimonidine exhibiting activity while clonidine isinactive.

[0097] B. Brimonidine, but not Clonidine or Tizanidine, AlleviatesSulprostone-Induced Hypersensitivity in the Absence of Sedation

[0098] Sedation limits the utility of many pharmaceuticals, includingα-2 agonists. The α-2 agonists were therefore compared to test whetherthere was a difference in the dose that resulted in alleviation ofsensory hypersensitivity relative to the dose that resulted in sedation.

[0099] For three α-2 agonists (tizanidine, clonidine and brimonidine),sedative effects and the ability to block tactile hypersensitivity werecompared at various doses in models of locomoter activity andsulprostone-induced tactile hypersensitivity, respectively. The tactilehypersensitivity of 5-6 mice per group was scored every five minutesbetween 15 and 50 minutes following intraperitoneal dosing. Vehicletreated animals typically had a score of about 4. In addition, thelocomoter activity of 5-6 mice per group was measured in a five minuteperiod 30 minutes following intraperitoneal dosing. The locomoteractivity relative to vehicle-treated animals was expressed as apercentage; percentage sedation was calculated as 100% minus the percentlocomoter activity. As shown in FIG. 6, of the three α-adrenergicagonists assayed, only brimonidine produced an analgesic effect that wasseparable from sedation. These results demonstrate that brimonidine isdistinct from other α-2 pan-agonists such as clonidine and tizanidine inthe ability to alleviate sympathetically-enhanced disorders such assulprostone-induced tactile hypersensitivity without concomitantsedation.

[0100] C. Variable α-2 Versus α-1 Functional Selectivity of α-AdrenergicPan-Agonists

[0101] The α-adrenergic receptor pharmacological profiles of brimonidineand clonidine were analyzed in assays using cell lines stably expressingα-2A, α-2C, α-1A and α-1B receptors.

[0102] Consistent with previous studies, the order of potency forinhibiting forskolin-induced cAMP accumulation in PC12 cells stablyexpressing either α-2A or α-2C receptor (FIGS. 7a, b; Table 2) wasdexmedetomidine, which was greater than or equal to brimonidine, whichwas greater than clonidine, which was greater than tizanidine, which wasgreater than or equal to phenylephrine (Jasper et al., Biochem.Pharmacol. 55:1035-1043 (1998); Pihlavisto et al., Eur. J. Pharmacol.385:247-253 (1999)). Brimonidine, clonidine and tizanidine wereapproximately 10-fold more potent at the α-2A receptor than the α-2Creceptor.

[0103] The same compounds were functionally tested for the ability tostimulate α-1-mediated increases in intracellular calcium in HEK293cells stably expressing the α-1A and α-1B receptor (FIGS. 7c, d; Table2). The order of potency at the α-1A and α-1B receptors wasphenylephrine, which was greater than clonidine, which was greater thantizanidine, which was equal to dexmedetomidine, which was greater thanbrimonidine. The α-2 agonists, clonidine, tizanidine anddexmedetomidine, were partial agonists while brimonidine exhibited weakactivity at the α-1A receptor and no activity at the α-1B receptor.Thus, although clonidine and tizanidine have previously beencharacterized as “α-2 selective” agonists in binding assays, thesecompounds display a less than 10-fold selectivity between α-2 and α-1receptor activation in functional assays. In contrast, dexmedetomidinewas approximately 300-fold selective in functional assays, andbrimonidine, the most highly selective compound in functional assays,exhibited greater than 1000-fold selectivity for α-2 receptors relativeto α-1 receptors (see Table 2). These results demonstrate thatbrimonidine is a highly selective α-2 versus α-1 agonist and that thedifferential α-2/α-1 selectivity of brimonidine contrasts with theselectivity of other pan-agonists such as clonidine.

[0104] The difference in α-2/α-1 selectivity between clonidine andbrimonidine indicates that the α-1 agonist activity of clonidine canaugment the increased sympathetic tone of the α-2C knockout mice andmask the analgesic activity of clonidine in the sulprostone model. Theseresults are supported by the ability of co-administration of the α-1antagonist prazosin with clonidine to restore the analgesic activity ofclonidine in α-2C knockout mice (FIG. 7e). Prazosin had no analgesicactivity by itself in wildtype or α-2C knockout mice.

[0105] In sum, these results indicate that the loss of clonidine, butnot brimonidine, analgesic activity in the α-2C knockout mice can be aresult of clonidine's α-1 agonist activity and that the α-1 agonistactivity of many “α-2 agonists” can limit their ability to treatstress-associated and other sympathetically-enhanced disorders.

[0106] Stable cell lines expressing an adrenergic receptor wereestablished as follows. The bovine α-1A, hamster α-1B, human α-2A andhuman α-2C receptor cDNAs were blunt-end subcloned into the NheI-EcORIsites in the retroviral vector pCL BABE Puro. The retroviral constructswere verified by double stranded DNA sequencing. High titer pseudotypedretroviral particles were produced by co-transfecting HEK293GP, a HEK293cell line stably expressing Gag-Pol of the Maloney leukemia virus, withthe appropriate retroviral vector and pMD.G, an expression vector forthe vesicular stomatitis virus envelope protein, VSV-G. Sixteen hoursafter transfection, the media (DMEM, 10% FCS) was changed; the hightiter (˜1×10⁶ pfu/mL) media was then harvested forty-eight hours later.The supernatant was filtered through a 0.4 uM filter.

[0107] The human α-2A and α-2C receptor supernatants were added, invarying amounts, to naive PC12 cells, which were then incubated for 48hours. The transduced cell populations were replated at a lower densityand grown in media containing 100 μg/ml puromycin. Non-transduced cellswere killed within three days, and single foci grew within two months.The foci were picked, expanded, and assayed for receptor density bybrimonidine radioligand binding. Functional α-2 receptor activity wasconfirmed by inhibition of forskolin-induced cAMP accumulation.

[0108] The bovine α-1A and hamster α-1B receptor supernatants wereadded, in varying amounts, to naive HEK293 cells, which were thenincubated for 48 hours. The transduced cell populations were replated ata lower density and grown in media containing 0.25 ug/ml puromycin.Significant cell death was evident within three days, with single fociappearing within two weeks. After the foci were picked and expanded,expanded subclones were functionally assayed for α-1 receptor expressionby measuring phenylephrine-induced intracellular Ca⁺² accumulation.Receptor density was measured in a prazosin radioligand binding assay.

[0109] Intracellular Ca⁺² responses were measured as follows in HEK293cells stably expressing either the bovine α-1A or hamster α-1Badrenergic receptor. Between 40,000 to 50,000 cells were plated per wellin 96-well poly-D-lysine coated plates in 0.2 ml DMEM containing 10%heat-inactivated fetal calf serum, 1% antibiotic- antimycotic and 0.25μg/ml puromycin one day prior to use. Cells were washed twice with HBSSsupplemented with 10 mM HEPES, 2.0 mM CaCl₂ and 2.5 mM probenicid, andsubsequently incubated at 37° C. for 60 minutes with 4 μM Fluo-4(Molecular Probes; Eugene, Oreg.). The extracellular dye was washed fromthe plates twice prior to placing the plates in the fluorometric imagingplate reader (FLIPR; Molecular Devices; Sunnyvale, Calif.). Ligands werediluted in HBSS and aliquoted into a 96-well microplate. Drugs weretested over the concentration range of 0.64 nM to 10,000 nM. Data forCa⁺² responses were obtained in arbitrary fluorescence units. TABLE 2Functional α-2 versus α-1 selectivity of α-adrenergic agonists humanα_(2A) human α_(2C) bovine α_(1A) hamster α_(1B) α_(1A)/ Compound EC₅₀ %E EC₅₀ % E EC₅₀ % E EC₅₀ % E α_(2A) Brimonidine 0.86 ± 0.1  91 8 ± 3 931132 ± 281  15 943 ± 247 12 1316 Dexmedetomidine 0.8 ± .01 93 0.48 ± .2 90 376 ± 97  59 364 ± 72  62 289 Clonidine 10 ± 1  94 56 ± 28 84 89 ± 1662 83 ± 10 63 8.9 Tizanidine 86 ± 35 93 1231 ± 376  85 264 ± 37  63 322± 31  61 3.1 Phenylephrine 306 ± 19  94 340 ± 131 87 9 ± 1 110 10 ± 1 110 .03

[0110] Intracellular cAMP measurement was performed as follows. PC12cells stably expressing the human α-2A or human α-2C adrenergicreceptors were plated in 96-well poly-D-lysine coated plates at adensity of 30,000 cells per well in 100 μl DMEM supplemented with 10%horse serum, 5% heat inactivated fetal bovine serum, 1%antibiotic-antimycotic and 100 μg/ml puromycin. Cells were grownovernight at 37° C. and 5% CO₂. Cells were dosed by adding an equalvolume of media containing IBMX (to a final concentration of 1 mM),forskolin (to a final concentration of 10 uM) and the appropriate drugdilution (to a final concentration of between 10⁻⁵ M and 10⁻¹² M). Aftera 10 minute incubation, the media was aspirated and the cells lysed with200 ul lysis buffer (Amersham Biosciences; Piscataway, N.J.). Plateswere stored at −20° C. for up to 24 hours prior to assay. IntracellularcAMP was determined using the Biotrak cAMP enzyme immunoassay system(Amersham Biosciences) according to the manufacturer's instructions.Plates were read on a plate reader at 450 nm.

[0111] Dose response curves for in vitro assays were generated usingKaleidaGraph (Synergy Software; Reading, Pa.) by least squares fits tothe equation, response=maximum response+((minimum response−maximumresponse)/(1+(concentration of ligand/EC₅₀)). The percent efficacy wasdetermined by comparing the maximum effect of the compound to the effectof a standard full agonist, which was phenylephrine for α-1 receptorsand brimonidine for α-2 receptors.

[0112] All journal article, reference and patent citations providedabove, in parentheses or otherwise, whether previously stated or not,are incorporated herein by reference in their entirety.

[0113] Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claims.

We claim:
 1. A method of preventing or reducing the severity of astress-associated condition in a subject, comprising systemicallyadministering to said subject an effective amount of brimonidine or apharmaceutically acceptable salt, ester, amide, sterioisomer or racemicmixture thereof, wherein said stress-associated condition is selectedfrom the group consisting of gastrointestinal disease; irritable bowelsyndrome (IBS); dyspepsia; tachycardia, provided that said tachycardiais not associated with myocardial ischemia; panic attack;insulin-resistance; type II diabetes; a non-inflammatory dermatogicalcondition; a disorder of muscle contraction; sensory hypersensitivityassociated with migraine; and a behavioral disorder.
 2. The method ofclaim 1, wherein said condition is gastrointestinal disease.
 3. Themethod of claim 1, wherein said condition is irritable bowel syndrome.4. The method of claim 1, wherein said condition is dyspepsia.
 5. Themethod of claim 1, wherein said condition is tachycardia, provided thatsaid tachycardia is not associated with myocardial ischemia.
 6. Themethod of claim 5, wherein said tachycardia is associated with apulmonary disorder.
 7. The method of claim 1, wherein said condition isa panic attack.
 8. The method of claim 1, wherein said condition isinsulin-resistance.
 9. The method of claim 1, wherein said condition istype II diabetes.
 10. The method of claim 1, wherein said condition is anon-inflammatory dermatological condition.
 11. The method of claim 1,wherein said condition is a disorder of muscle contraction.
 12. Themethod of claim 11, wherein said disorder of muscle contraction is adisorder of skeletal muscle contraction.
 13. The method of claim 11,wherein said disorder of muscle contraction is a disorder of smoothmuscle contraction.
 14. The method of claim 13, wherein said disorder ofsmooth muscle contraction is associated with cystitis.
 15. The method ofclaim 13, wherein said disorder of smooth muscle contraction isassociated with non-bacterial prostatitis.
 16. The method of claim 11,wherein said disorder of muscle contraction is associated with tensiontype headache.
 17. The method of claim 1, wherein said condition issensory hypersensitivity associated with migraine.
 18. The method ofclaim 1, wherein said condition is a behavioral disorder.
 19. The methodof claim 1, wherein said effective amount is administered orally. 20.The method of claim 1, wherein said effective amount is administeredtopically.
 21. The method of claim 1, wherein said effective amount isadministered via a patch.
 22. The method of claim 1, wherein saideffective amount is administered intravenously.